This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. COBRE Project 1 will study the molecular physiology and regulation of volume-regulated anion channels in the heart. The molecular mechanism of Cl- channel regulation is an important issue for understanding how cells regulate their volume and for understanding structure/function relationships of ion channel proteins in general. We previously proposed ClC-3, a member of the ClC superfamily of voltage-dependent Cl- channels, as a molecular candidate responsible for native volume-regulated outwardly rectifying anion channels (VSOACs) in cardiac and smooth muscle cells. The experiments proposed in this project will provide new insights into the role of phosphorylation by various protein kinases of amino terminus amino acids in the regulation of the two major ClC-3 isoforms, use transgenic mice to further examine the relationship between ClC-3 and native VSOACs and reveal their physiological role, and finally will use molecular and proteomic approaches to identify the major components and accessory proteins that constitute the native VSOAC multimeric protein complex. Since the activation of cardiac Cl- channels can produce significant effects on action potential duration and automaticity, and are key regulators of cell volume homeostasis, these channels have important clinical significance for several myocardial diseases, including cardiac arrhythmias, myocardial ischemia, congestive heart failure and hypertrophy. This project has significant potential of elucidating the normal physiological and possible pathophysiological role of volume-sensitive Cl- channels in the heart and cardiovascular system.